Dual promoters control expression of the Bacillus anthracis virulence factor AtxA

Abstract

The AtxA virulence regulator of Bacillus anthracis is required for toxin and capsule gene expression. AtxA is a phosphotransferase system regulatory domain-containing protein whose activity is regulated by phosphorylation/dephosphorylation of conserved histidine residues. Here we report that transcription of the atxA gene occurs from two independent promoters, P1 (previously described by Dai et al. [Z. Dai, J. C. Sirard, M. Mock, and T. M. Koehler, Mol. Microbiol. 16:1171-1181, 1995]) and P2, whose transcription start sites are separated by 650 bp. Both promoters have -10 and -35 consensus sequences compatible with recognition by sigma(A)-containing RNA polymerase, and neither promoter depends on the sporulation sigma factor SigH. The dual promoter activity and the extended untranslated mRNA suggest that as-yet-unknown regulatory mechanisms may act on this region to influence the level of AtxA in the cell.

FIG. 1.

pXO1-118-atxA region on the pXO1 virulence plasmid: restriction map of the pXO1 plasmid carrying the pXO1-118 and atxA genes with the restriction sites relevant for this study. The open arrows indicate the direction of gene transcription. The fragments cloned in the plasmids are indicated below the restriction map. The directions of transcription of the spectinomycin resistance cassette used for gene deletion are indicated by arrowheads. The positions of the oligonucleotide primers used in the RT-PCR and primer extension reactions are indicated above the restriction map. The designations of the primers are shortened for clarity.

FIG. 2.

PA expression in the pXO1-118 gene deletion strain. (A) β-Galactosidase assay of the pagA-lacZ reporter in parental strain 34F2 (•) and in the 34F2Δ118 mutant strain (▴). Cells were grown in LB medium containing kanamycin. Time zero is the time of transition between logarithmic growth and the stationary phase. (B) Western blot analysis of the PA levels in culture supernatants of parental and 34F2Δ118 mutant strains complemented with the pXO1-118 gene on the multicopy vector pHT315. Samples were collected at the time point indicated by the double arrow in panel A. Lane 1, 34F2/pHT315; lane 2, 34F2/pAtxA3; lane 3, 34F2Δ118/pHT315; lane 4, 34F2Δ118/pAtxA3; lane 5, purified PA (0.01 μg). Lane M contained Western blot standard Magic Mark XP (Invitrogen). The molecular masses of bands (in kDa) are indicated on the left. The bands were quantitated with the Image Quant software (Amersham-Molecular Dynamics), and the pixel value for each lane is indicated below the gel.

FIG. 3.

Transcriptional analysis of atxA-lacZ fusion constructs in parental strain 34F2. Cultures used for β-galactosidase assays were grown in LB medium, and samples were taken at hourly intervals before and after the transition (time zero) from exponential phase to stationary phase. Symbols: +, pTCVlac; □, pAtxA10; ⋄, pAtxA12; ○, pAtxA13; ▿, pAtxA15; ▪, pAtxA18; ⧫, pAtxA19; ▴, pAtxA20; •, pAtxA21; ▵, growth curve for a representative strain (34F2/pAtxA15). OD₅₂₅, optical density at 525 nm.

FIG. 4.

RT-PCR analysis of the atxA mRNA. RT-PCR was carried out with mRNA extracted from strain 34F2 and treated with RNase-free DNase. The mRNA was first incubated with (+) or without (−) RT and oligonucleotide primer AtxART2 (RT2 in Fig. 1). An aliquot of the reaction mixture was then incubated with oligonucleotide primers AtxART2 and AtxART3 (RT3 in Fig. 1) (lanes 1 and 2) or oligonucleotide primers AtxART2 and AtxA5′upEcoRI (5′up in Fig. 1) (lanes 4 and 5). Lanes 3 and 6 contained the products of PCR amplifications carried out with oligonucleotides AtxART2 and AtxART3 and with oligonucleotides AtxART2 and AtxA5′upEcoRI, respectively, using 34F2 genomic DNA as the template. Lane M contained the 1 Kb Plus DNA ladder (New England Biolabs). Molecular sizes (in base pairs) are indicated on the right.

FIG. 5.

Mapping the P2 5′ end of the atxA mRNA transcript. (A) Sequencing reactions (lanes G, A, T, and C) were carried out with plasmid pAtxA12 using oligonucleotide primer AtxART4, and the mixtures were run on a 4% acrylamide-8 M urea gel alongside the mixture for a primer extension reaction carried out with the same oligonucleotide (lane RT4). The nucleotide sequence surrounding the 5′ ends (indicated by an asterisk) of the noncoding strand is shown. (B) Nucleotide sequence of the pXO1 plasmid region containing the pXO1-118 gene and the pXO1-118-atxA intergenic region. The ATG start codons for atxA and the pXO1-118 gene are enclosed in boxes, and the open arrows indicate the direction of transcription. The stop codons for the pXO1-118 gene are also indicated. Putative −10 and −35 sequences are underlined. The region protected by AbrB in DNase footprinting analysis (30) is indicated by overlining.

FIG. 6.

Western blot analysis of protein levels in parental strain 34F2 and the 34F2ΔsigH strain. Samples were collected at the mid-exponential phase (lanes 1, 4, and 7), transition phase (lanes 2, 5, and 8), and stationary phase (lanes 3, 6, and 9). Lanes 1 to 3 contained samples from parental strain 34F2; lanes 4 to 6 contained samples from the sigH mutant strains; and lanes 7 to 9 contained samples from the abrB mutant strains. Lane M contained the Magic Mark XP standard (Invitrogen); the molecular mass of the smaller band in this marker is 20 kDa, and that is why no band is present in this lane in the AbrB panel. Lane H contained purified B. anthracis SigH (0.05 μg). Lane A contained purified B. anthracis AtxA protein (0.006 μg). Lane P contained purified B. anthracis PA (0.1 μg). Lane B contained lysate of the B. subtilis JH642 parental strain as a positive control for AbrB (10.6 kDa). The sizes (in kDa) of the bands for the Magic Mark XP standard visible in each panel are indicated on the right.

FIG. 7.

Transcription analysis of the atxA promoter in the sigH mutant strain. β-Galactosidase assays were carried out using LB medium (A) or R medium under CO₂/bicarbonate growth conditions (B), and samples were removed at hourly intervals. (A) Strain 34F2ΔsigH carrying the following plasmids: pTCVlac (+), pAtxA10 (□), pAtxA12 (⋄), pAtxA13 (○), pAtxA15 (▿), pAtxA19 (⧫), pAtxA20 (▴), and pAtxA21 (•). ▵, representative growth curve (pAtxA10). (B) Direct comparison of pAtxA10 and pAtxA12 transcription in parental strain 34F2 and the sigH mutant strain grown in R medium. Symbols: □, 34F2/pAtxA10; ▪, 34F2ΔsigH/pAtxA10; ⋄, 34F2/pAtxA12; ⧫, 34F2ΔsigH/pAtxA12; ▵, representative growth curve (34F2/pAtxA10). OD₅₂₅, optical density at 525 nm.